Molecular Mechanisms of Plant Responses to Copper: From Deficiency to Excess

Copper (Cu) is an essential nutrient for plant growth and development. This metal serves as a constituent element or enzyme cofactor that participates in many biochemical pathways and plays a key role in photosynthesis, respiration, ethylene sensing, and antioxidant systems. The physiological significance of Cu uptake and compartmentalization in plants has been underestimated, despite the importance of Cu in cellular metabolic processes. As a micronutrient, Cu has low cellular requirements in plants. However, its bioavailability may be significantly reduced in alkaline or organic matter-rich soils. Cu deficiency is a severe and widespread nutritional disorder that affects plants. In contrast, excessive levels of available Cu in soil can inhibit plant photosynthesis and induce cellular oxidative stress. This can affect plant productivity and potentially pose serious health risks to humans via bioaccumulation in the food chain. Plants have evolved mechanisms to strictly regulate Cu uptake, transport, and cellular homeostasis during long-term environmental adaptation. This review provides a comprehensive overview of the diverse functions of Cu chelators, chaperones, and transporters involved in Cu homeostasis and their regulatory mechanisms in plant responses to varying Cu availability conditions. Finally, we identified that future research needs to enhance our understanding of the mechanisms regulating Cu deficiency or stress in plants. This will pave the way for improving the Cu utilization efficiency and/or Cu tolerance of crops grown in alkaline or Cu-contaminated soils.


Introduction
Copper (Cu) is an essential element for living organisms and plays an important role in numerous biological processes.In humans, Cu acts as a cofactor for a variety of enzymes, such as Cu/zinc (Zn) superoxide dismutase (Cu/Zn-SOD, CSD), cytochrome c oxidase, mitogen-activated protein kinase MEK1, and CAMP-degrading phosphodiesterase PDE3B.Insufficient Cu intake in humans can lead to Cu deficiency disorders such as anemia, pancytopenia, and ataxia [1].Chronic Cu poisoning occurs when humans are exposed to excessive amounts of Cu for a long period of time.Cu toxicity often presents with the development of cirrhosis, accompanied by hemolytic attacks and damage to the renal tubules, brain, and other organs [2].Cu is one of the 17 essential elements required for plant growth.Plants have low requirements for this element; however, similar to other essential nutrients, it is crucial for their growth.Under physiological conditions, Cu participates in various cellular metabolic processes [3].Despite its importance in metabolic activity, little research has been conducted on its role in plant homeostasis.Although Cu is abundant in most soils, specific soil pH and redox conditions significantly influence the concentration of Cu.Cu deficiency in plants typically occurs in calcareous alkaline soils with a high pH or soils with a high organic content, which can result in severe nutritional disorders [4,5].Although Cu deficiency can be addressed using Cu-based fertilizers, this approach is not environmentally friendly and may result in the accumulation of toxic Cu in the soil [6].Cu availability is generally higher in acidic soils, which increases its potential toxicity to plants [7].Owing to prolonged industrial activities and the widespread use of Cu-containing fungicides in agricultural production, a significant amount of Cu is released into the soil.This influx of Cu may be a key factor contributing to Cu toxicity in plants [8].It has been reported that the application of certain soil amendments can alter the soil pH, thereby influencing the bioavailability of Cu in the soil [9].The application of lime can promote the alkalization of acidic soil solutions, thus inducing the deprotonation of acid functional groups on the surface of soil particles, increasing the cation exchange capacity of the soil, and enhancing the adsorption of Cu [10].However, this is a temporary solution.Owing to the potential leaching of Cu along soil profiles, the increased mobility of Cu may pose an environmental risk.Some researchers have applied fly ash to Cu-polluted soil to increase the soil pH and reduce the uptake of Cu by plants [11].However, this strategy is costly and often impractical for farmers with poor-quality land.
In response to changes in external Cu levels, plants must either efficiently acquire and use Cu under limited conditions or detoxify the metal with excess supply.Plants have evolved complex molecular regulatory mechanisms to maintain Cu homeostasis and ensure normal life activities during long-term environmental adaptation [12].To date, various Cu transporters, chaperones, and chelators have been identified to be involved in Cu uptake, transport, and maintenance of cellular homeostasis in plants.This review discusses the functions of Cu-transport-related proteins in various plant species.Table 1 lists the Cu transporters discussed in the article.

Table 1.
Copper uptake and transport genes in different species.

Gene Subcellular Localization
Cu plays a crucial role in various processes of the plant life cycle, such as photosynthesis, respiration, ethylene sensing, clearance of reactive oxygen species (ROS), cell wall metabolism, and hormone signaling [48,49].As a redox-active metal, Cu can cycle between the reducing state Cu 1+ and the oxidizing state Cu 2+ under physiologically relevant conditions, thereby catalyzing single-electron transfer reactions [50].The reduced form of Cu 1+ preferentially binds sulfur-containing compounds with mercaptan or thioether groups, whereas the oxidized form of Cu 2+ mainly coordinates with the oxygen or imidazole nitrogen groups [51,52].Therefore, Cu acts as a structural or catalytic cofactor in essential cellular enzymes that take advantage of its redox potential.
It is estimated that 30% of Cu is present in the chloroplasts of green plants [53].However, 60-80% of Cu in chloroplasts is present in thylakoids, indicating the important role of Cu in photosynthetic proteins and complexes [53,54].Plastocyanin (PC), a small soluble Cu protein in the thylakoid lumen, plays a crucial role in both linear and cyclic photosynthetic electron transport and is essential for the photoautotrophic growth of plants [55].PC is one of the most abundant proteins in the thylakoid membrane.It was first isolated from Chlorella and has since been found in cyanobacteria, algae, and plant chloroplasts [55].During photosynthesis, PC serves as an electron carrier from cytochrome f (Cyt f ) in the cytochrome b 6 f complex (Cyt b 6 f ) to P 700 in photosystem I (PSI) [56,57].Some studies have shown that Chlamydomonas can use heme-containing cytochrome c 6 (Cyt c 6 ) to functionally replace PC when Cu is limited [58].However, in terrestrial plants, the modified form Cyt c 6A of Cyt c 6 is not suitable for use as an electron carrier between cytochrome b6f and PSI because of its unique topological structure; therefore, it cannot replace PC in Arabidopsis [59,60].Two PC subtypes are encoded in most angiosperms, including Arabidopsis [61], poplar [62], tobacco [63], rice [64], and Physcomitrella patens [65].These two subtypes have essentially the same function in terms of electron transport activity.However, in Arabidopsis, one protein subtype (PC1) accumulates at lower levels, whereas the second subtype (PC2) is more abundant but also more sensitive to Cu availability at the protein level [66,67].Polyphenol oxidase (PPO), a Cu-rich enzyme, is found in the thylakoid cavity of many plants (such as poplar and spinach).It catalyzes the conversion of monophenol to o-dihydroxyphenyl and o-quinone, which easily leads to black or brown pigmentation.It is thought to play a role in defense against attacks by herbivores or pathogens [68][69][70].
Cu serves as a cofactor for the terminal enzyme complex cytochrome c oxidase (COX, or Complex IV) in the mitochondrial respiratory chain [71,72].The cytochrome c reductase complex (Complex III) transfers electrons from panthenol to cytochrome c.Finally, COX accepts electrons from cytochrome c, facilitating the conversion of oxygen and H into water [73].Eukaryotic COX has 11-13 subunits, with the three largest subunits (COX1, COX2, and COX3) encoded by the mitochondrial DNA [74][75][76].The normal function of COX requires five types of cofactors: two hemes, three Cu ions and Zn, manganese (Mn), and sodium (Na) ions [76].
Multicopper oxidase (MCO) is a superfamily of proteins present in a wide variety of organisms that binds to four Cu ions [77,78].In plants, MCO consists mainly of ascorbate oxidase and laccase [78].Ascorbate oxidase is found in exosomes and plays an important role in cell expansion, salt tolerance, and plant productivity [79,80].Laccase (LAC), similar to ascorbate oxidase, is present in exosomes and uses molecular oxygen as the final electron acceptor to catalyze the oxidation of various substrates (such as phenols, aromatics, or aliphatic amines) to generate reactive radicals.These radicals then undergo non-enzymatic oxidation or reduction reactions to produce water and oligomers [81].LAC plays an important role in various physiological and biochemical processes, including lignin and flavonoid biosynthesis [82,83], pigmentation [84], maintenance of the cell wall structure and integrity [82], and salt stress tolerance [85].Another Cu protein found in exosomes is plantacyanins, which belong to the phytocyanin family of blue Cu proteins and play a role in guiding pollen tubes [86].
CSD is a Cu protein and one of the three common types of superoxide dismutase (SOD) in plants: CSD, Mn-SOD, and Fe-SOD [87].CSD can catalyze the conversion of superoxide free radicals into molecular oxygen and hydrogen peroxide, which play a crucial role in scavenging ROS and alleviating oxidative and abiotic stress [88].Three subtypes of CSD have been identified in Arabidopsis thaliana: CSD1 in the cytoplasm, CSD2 in the chloroplast, and CSD3 in the peroxisome [89].Evidence suggests that the Cu companion of superoxide dismutase (CCS) is a key factor in the integration of Cu into CSD in eukaryotes [90,91].
Ethylene signaling is another Cu-dependent process [92].Cu is a cofactor for ethylene receptors.Ethylene affects many aspects of plant life, including seed germination, stem thickening, fruit ripening, stomatal opening, and plant responses to biotic and abiotic stresses [92][93][94].Ethylene is sensed by a series of membrane-binding receptors that are negative regulatory molecules in the ethylene reaction.When the receptors bind to ethylene, their activation response signaling is turned off [95].
Cu is also involved in the synthesis of molybdenum cofactors [96], the biosynthesis of quinones and carotenoids [97,98], pollen formation [99], and oxidative phosphorylation [98].Cu can also enhance the flower and vegetable yields, flavor, color, sugar content, and storage life of fruits [100][101][102].Moreover, Cu ions are widely used in agriculture owing to their antibacterial, antiviral, and insect-resistant properties [103,104].

Cu Dynamics in Soil
Cu is a reddish-brown transition metal with a natural abundance of 60 mg/kg in Earth's crust [105].The natural form of Cu usually exists in two forms: the reduced cuprous ion, Cu (I), and the Cu oxide ion, Cu (II) [106].In soil solutions, Cu can exist in its free form, such as Cu 2+ .However, in most cases, Cu is complexed with inorganic anionic binders or organic molecules.Approximately 80% of Cu in soil exists in the form of insoluble compounds (oxides and sulfides), which are not conducive to plant utilization [9].In contrast, approximately 20% of Cu in the soil is in the soluble form (hydroxyl and carbonate), which is typically available to plants.However, the bioavailability of soluble Cu depends on the functional groups of the active soil particles and their adsorption capacity, which are influenced by various factors [107] (Figure 1).It is generally believed that a soil Cu content <8 mg/kg may cause Cu deficiency symptoms in crops [108].Varying degrees of Cu deficiency exist in agricultural soils in many areas of the world, adversely affecting the yields of rice, wheat, and corn [109][110][111][112][113].
Cu is also involved in the synthesis of molybdenum cofactors [96], the biosynthesis of quinones and carotenoids [97,98], pollen formation [99], and oxidative phosphorylation [98].Cu can also enhance the flower and vegetable yields, flavor, color, sugar content, and storage life of fruits [100][101][102].Moreover, Cu ions are widely used in agriculture owing to their antibacterial, antiviral, and insect-resistant properties [103,104].

Cu Dynamics in Soil
Cu is a reddish-brown transition metal with a natural abundance of 60 mg/kg in Earth's crust [105].The natural form of Cu usually exists in two forms: the reduced cuprous ion, Cu (I), and the Cu oxide ion, Cu (II) [106].In soil solutions, Cu can exist in its free form, such as Cu 2+ .However, in most cases, Cu is complexed with inorganic anionic binders or organic molecules.Approximately 80% of Cu in soil exists in the form of insoluble compounds (oxides and sulfides), which are not conducive to plant utilization [9].In contrast, approximately 20% of Cu in the soil is in the soluble form (hydroxyl and carbonate), which is typically available to plants.However, the bioavailability of soluble Cu depends on the functional groups of the active soil particles and their adsorption capacity, which are influenced by various factors [107] (Figure 1).It is generally believed that a soil Cu content <8 mg/kg may cause Cu deficiency symptoms in crops [108].Varying degrees of Cu deficiency exist in agricultural soils in many areas of the world, adversely affecting the yields of rice, wheat, and corn [109][110][111][112][113]. The adsorption capacity of Cu increased with higher levels of clay minerals, iron (Fe), aluminum (Al), Mn hydroxide or oxygen hydroxides, carbonate, and organic matter [114,115].Soil pH, cation exchange capacity, and soil organic matter (SOM) content are important factors in regulating the conversion between the solubility, adsorption, desorption, and form of heavy metals (including Cu) and, therefore, the availability of Cu [49].It has been reported that the available Cu content in the soil is significantly positively correlated with soil pH [116].When the soil pH decreases, the Cu adsorbed or co-precipitated by carbonate can easily be re-released into the environment.Additionally, some evidence suggests that plants can alter the pH of the soil around their roots, thereby altering the bioavailability of metals in the root environment [117,118].For example, plant root exudates (e.g., citric acid and malic acid) can cause the rhizosphere pH to be more acidic than that of bulk soil, leading to the dissolution of Cu, which may then be absorbed near the The adsorption capacity of Cu increased with higher levels of clay minerals, iron (Fe), aluminum (Al), Mn hydroxide or oxygen hydroxides, carbonate, and organic matter [114,115].Soil pH, cation exchange capacity, and soil organic matter (SOM) content are important factors in regulating the conversion between the solubility, adsorption, desorption, and form of heavy metals (including Cu) and, therefore, the availability of Cu [49].It has been reported that the available Cu content in the soil is significantly positively correlated with soil pH [116].When the soil pH decreases, the Cu adsorbed or co-precipitated by carbonate can easily be re-released into the environment.Additionally, some evidence suggests that plants can alter the pH of the soil around their roots, thereby altering the bioavailability of metals in the root environment [117,118].For example, plant root exudates (e.g., citric acid and malic acid) can cause the rhizosphere pH to be more acidic than that of bulk soil, leading to the dissolution of Cu, which may then be absorbed near the roots [117,119].In addition to soil pH, SOM content significantly influences the fluidity and bioavailability of heavy metals in the soil [120].SOM is the main adsorbent that controls the distribution of heavy metals in soil solids and solutions.Its main components are humic and fulvic acid, which are organic compounds with complex structures [121].These structures include alkyl, carbonyl, sugar residues, heteroatoms, and various other functional groups [121].The complexation of heavy metals with organic ligands affects their mobility by either increasing or decreasing their adsorption onto the mineral surfaces [122,123].Cu and SOM form strong complexes; therefore, Cu-SOM complexes are usually the dominant form present in soils with a high SOM content [124].Owing to the high affinity of SOM for Cu, the effect of soil pH on the fluidity and availability of Cu diminishes significantly when SOM content is high [125].Therefore, plants growing in swampy, humus, calcareous, or alkaline soils with high organic matter content in arid and semi-arid environments may develop Cu deficiencies.
In addition to the physical and chemical properties of the soil, industrial production and human activities are important factors affecting the bioavailability of Cu in the soil (Figure 1).Industrial production includes factories that manufacture or use Cu metal or related compounds, Cu mining and drainage, fertilizer production, and chemical manufacturing.These activities often lead to the release of high levels of Cu into the soil [126,127].It is estimated that 939,000 tons of Cu have been discharged from the natural environment worldwide over the past 50 years [128].It has been reported that the Cu content in Chinese soil ranges from 0.3 to 272 mg/kg, with an average of 21.9 mg/kg [129].However, in some seriously polluted areas, such as near mining sites, the Cu content in the soil can reach levels as high as 5000 mg/kg.When the Cu content in the soil exceeds 50-250 mg/kg, it can negatively affect plant growth [129].Furthermore, other anthropogenic activities associated with agricultural practices, especially the application of Cu-based agrochemicals such as fertilizers, pesticides, fungicides, and herbicides, are major sources of Cu deposition in soil [130].For example, to prevent citrus canker, some citrus growers often use Cu-containing fungicides such as Bordeaux liquid, Cu hydroxide, Cu oxychloride, and thiazide Cu, as well as excessive application of Cu-containing trace element fertilizers.This practice leads to the annual accumulation of Cu on the surface of the orchard soil, consequently increasing Cu levels in both soil and trees [131,132].Moreover, some fruit farmers unilaterally pursue economic benefits by reducing the input of organic fertilizer and applying a high content of acid compound fertilizer, along with undecomposed livestock and poultry manure, leading to soil acidification [133].Therefore, excess available Cu is common in citrus orchard soils.Research has continued to develop effective remediation methods to treat contaminated land and mitigate the negative impacts of Cu infiltration into the ecosystem.However, the choice and practicality of remediation measures depend on various factors, such as availability, cost, and duration of effectiveness.For example, farmers can minimize the toxicity of Cu to plants by using soil amendments (such as adding biochar, lime, and phosphate) to reduce the availability of Cu in the soil [108].However, the use of these soil amendments can increase farming costs, reduce herbicide effectiveness, and introduce other pollutants that have potentially negative effects on soil bacteria and animals.

Cu Deficiency
In plants, Cu deficiency typically manifests as a potential disease that can ultimately affect gene expression changes that regulate various biological processes as well as root and leaf morphology [134][135][136].The average Cu content in the plant dry weight is approximately 10 µg g −1 /DW, and the critical Cu deficiency is generally between 1 µg g −1 and 5 µg g −1 /DW [135,137,138].In plants, Cu deficiency leads to growth retardation, increased senescence, and reduced productivity [98,136,139].This may be caused by damage to the electron transport chain, leading to disruptions in photosynthesis and respiration [97].This can also result in reduced plastoquinone synthesis, unsaturated C 18 fatty acid content, isopropyl lipid synthesis, decreased CO 2 fixation, and sensitivity to environmental stress [140,141].Photosynthesis is the primary target of Cu deficiency [97].Cu deficiency leads to a reduction in plastocyanin formation, which affects PSI electron transport, thereby reducing the net photosynthetic rate [53,141].It has been suggested that severe Cu deficiency in plants can cause the disintegration of PSII complexes, directly altering the thylakoid structure and promoting the degradation of pigments (chlorophyll and carotenoid) [136,140,142].Additionally, because CSD function is severely limited by Cu deficiency, it may lead to oxidative stress [143,144].Owing to the poor migration rate of Cu in the phloem, the typical symptoms of Cu deficiency first appear in young leaves.These symptoms manifest as chlorosis at the top of the young leaves, leaf edge curling, leaf deformity, or even necrosis [98,139].
In Arabidopsis, Cu deficiency reduces the expression of genes involved in early anther development, leading to abnormal anther development and delayed flowering [99,145].Furthermore, a variety of crop species, such as wheat, rice, oats, barley, sweet corn, and sunflower, have been used to demonstrate that Cu deficiency has a greater effect on reproduction than on vegetative growth, leading to reduced grain/seed yields [98].Decreased fertility in Cu-deficient crops results from multiple factors, including stunted stigmas, reduced pollen fertility, and reduced pollination capacity [36].However, compared with other micronutrients (including Fe, Mn, and Zn), there are few studies on the effects of Cu deficiency on plant responses to environmental stresses such as temperature, drought, and pathogen invasion (Figure 1).

Cu Toxicity
Owing to the redox properties of Cu ions, excess Cu accumulated in cells can have a toxic effect on plants.Toxic Cu concentrations vary significantly depending on plant species and genotypes, leading to significant differences in Cu tolerance within and among species [146,147].Evidence suggests that photosynthesis is an early target of Cu toxicity [148].After exposure to Cu stress, plants often exhibit a decrease in chlorophyll content and net photosynthetic rate [149,150].The reduction in chlorophyll biosynthesis may be related to the structural damage of the photosynthetic apparatus on the thylakoid membrane under Cu stress and the interference of Cu on chlorophyll organization [151,152].Some researchers have found that Cu has an inhibitory effect on both photosystems on the thylakoid membrane [8,153,154].However, compared with PSI, PSII is more sensitive to Cu toxicity [8,153,154].High concentrations of Cu can damage the non-heme type Fe 2+ and cytochrome b 559 on the PSII acceptor side, as well as the Mn cluster and external proteins of the oxygen evolution complex on the donor side.This damage can ultimately lead to impaired electron transfer and a reduced photosynthetic rate [155][156][157][158]. High concentrations of Cu can lead to structural damage to thylakoids, alter their lipid composition [159], induce lipid peroxidation [154], inhibit photophosphorylation [160], and reduce the efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase [161].
As a redox-active metal, excess Cu induces an increase in cellular ROS concentrations, such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 •− ), and hydroxyl radicals ( • OH), and causes oxidative stress directly or indirectly by affecting cellular metabolic processes [162].Evidence suggests that lipid peroxidation is an indicator of oxidative stress in plants [163].In cells, the membrane system is the primary target of heavy metal poisoning.Excessive Cu also alters membrane lipids, affecting their total quantity, composition, and saturation levels [164].In addition, ROS generated by Cu stress can attack hydrogen atoms in fatty acid chains (such as linoleic acid) in plasma and organelle membranes, leading to the generation of aldehydes and lipid free radicals [165,166].These reactions can cause membrane lipid peroxidation, increased membrane permeability, massive extravasation of cellular contents, and cell death [167].Some evidence suggests that, although H 2 O 2 and O 2 •− can initiate lipid peroxidation, only • OH is reactive enough to cause lipid peroxidation in the presence of Fe and Cu [168].
Another common effect of Cu toxicity on plants is the reduced absorption and homeostasis of other mineral nutrients [146].Some evidence suggests that exposure to high concentrations of Cu reduces plant uptake of macronutrients, such as calcium (Ca), potassium (K), magnesium (Mg), and phosphorus (P), and trace elements, such as Fe, Mn, or Zn, or alters the distribution of these elements in roots and shoots [169][170][171][172]. High concentrations of Cu can disrupt the normal functions of certain ions during cell metabolism.Some researchers believe that Cu competes with the binding of -SH residues, thereby replacing cofactors (such as Mg 2+ , Zn 2+ , or Fe 2+ ) in photosynthetic pigments or proteins in chloroplasts.Cu alters the ionic composition of photosynthetic pigments or proteins, making them unstable or inactive, leading to their degradation, and consequently affecting photosynthetic activity [49].For example, Cu ions replace Mg 2+ in chlorophyll molecules, resulting in a reduction in the chlorophyll content [173].Excess Cu also reduces nitrate absorption, thereby inhibiting the growth of rice seedlings [174].
Symptoms of Cu toxicity vary widely among plant species and depend on the Cu content, duration, and developmental and physical stages of the plant [146,147].Seed germination is the most sensitive physiological process to Cu stress in plants.Even a low dose of Cu can reduce the seed germination rate, inhibit bud growth, and impede root formation [175,176].During plant growth, the root system plays an important role in absorbing nutrients from the soil and in responding to abiotic stress [177].Therefore, the response of plants to Cu toxicity first occurs in the roots.High concentrations of Cu in the soil inhibit and damage root growth, leading to reduced absorption of nutrients and moisture by the roots.Under Cu stress, the typical symptoms of roots include root discoloration, reduced root hair proliferation, reduced primary root elongation, and severe deformation of the root structure [49].Some evidence suggests that reduced root growth is often associated with the rupture of epidermal cells and root cell membranes and the thickening and rupture of the root cuticle [178,179].However, the mechanism by which Cu stress affects root physiological processes to inhibit root development and the recognition of Cu stress signals remain unclear.It has been suggested that Cu ions may affect root growth and development by altering root meristem cell viability and mitotic activity, regulating plant hormones such as auxin and cytokinin, and influencing lignin deposition in the root system [180].
Another common and major symptom of plant Cu toxicity is leaf chlorosis, which often results in milky or white spots or lesions [181].As Cu exposure increases, necrotic areas appear at the leaf tips and edges, whereas leaf area, diameter, and length decrease [182,183].In acute Cu poisoning, leaves may wilt before eventually becoming necrotic [183].Furthermore, Cu toxicity ultimately leads to reduced plant biomass and grain yields, which may pose serious health risks to humans through bioaccumulation in the food chain [7,149,184] (Figure 1).

Cu Transport in Plants
Cu is an essential micronutrient for plant growth and development, and its absorption by plants is an active transport process [185].The cell wall is the first barrier for Cu ions to enter plant root cells and contains many macromolecular substances (including amino, phosphoric acid, hydroxyl, carboxyl, and hydroxyl) [186].These coordination groups can undergo various physical or chemical reactions with positively charged Cu ions, adsorb excess Cu ions on the cell wall, and inhibit the entry of Cu ions into protoplasts.This process reduces the toxic effects of excessive Cu ions on plant cells [186,187].
Cu passes through the cell wall and forms various complexes with different amino acids and proteins within the cell.It is then transported to various tissues and organs through the xylem and phloem.At the tissue level, Cu transport mainly involves root absorption, vacuole sequestration, xylem and phloem loading, and node distribution and redistribution [188,189].At the cellular level, the Cu transporters identified in plants can be divided into two categories: those responsible for transporting extracellular Cu to the intracellular space (absorption transporters) and those responsible for transporting intracellular Cu to the extracellular space or organelles (efflux transporters) [190].In plant cells, Cu is maintained in a relatively balanced state through the coordinated regulation of these two types of Cu transporters.This balance ensures normal growth and development of plants and avoids the symptoms of Cu poisoning [188,191].To date, several Cu transporters, including the high-affinity Cu absorption transporters CTR/COPT, P1B-ATPase/heavy metal ATPase, zinc-iron transporter ZIP, yellow stripe-like protein YSL, and Cu chaperones, have been identified and functionally characterized in monocotyledonous and dicotyledonous plants [192].
The COPT family of transporters consists of three transmembrane domains (TMDs) that contain His and/or Met domains, and the conserved motifs MXXXM and GXXXXG are associated with TMD2 and TMD3 [193].According to the number of domains, the six COPT members reported in Arabidopsis thaliana can be divided into three categories.The first class consists of three Met-and His-rich domains: AtCOPT1, AtCOPT2, and AtCOPT6.These proteins exhibit strong affinity and transport activity for Cu, which is considered the primary reason for the ability of Cu to enter and exit cells [13].The second type of members is AtCOPT3 and AtCOPT5, which have low binding capacities for Cu and cannot effectively transport Cu ions.Interestingly, the third class is AtCOPT4, which lacks the Met residues and the MXXXM motifs required for Cu ion transport.However, this protein can assist in Cu transport in plants by interacting with other COPT proteins [194].P 1B -ATPase, also known as heavy metal-transporting ATPase (HMA), consists of eight genes in Arabidopsis thaliana (AtHMA1-AtHMA8) and nine genes in rice (OsHMA1-AtHMA9) [195].The HMA family includes members of all organisms.This class of proteins is not specific to metals, so the family can be divided into two subclasses: Cu subclass P 1B -ATPase and Zn subclass P 1B -ATPase [195].The Cu subclasses in Arabidopsis include AtHMA5-AtHMA8, whereas they consist of OsHMA4-OsHMA9 in rice.
ZIP protein family members are primarily responsible for the transport of metal ions (including Zn, Cu, Fe, Mn, and Cd) into the cytoplasm and play an important role in maintaining the ionic balance of the cell [196].At present, >100 ZIP proteins have been identified and are widely distributed in fungi, bacteria, animals, and plants; however, the functions of most genes have not been studied.Arabidopsis contains 11 ZIP (AtZIPl-AtZIP11) transporters, whereas rice contains 17 ZIP (OsZIP1-OsZIP17) transporters [25].
The YSL protein family primarily participates in the transport of metal elements in grasses, facilitating long-distance transport of nicotianamine (NA) and iron carriers within plants [197].Eight members of the YSL family (AtYSL1-AtYSL8) have been identified in Arabidopsis, and 18 members of rice (OsYSL1-OsYSL18).
Cu chaperones are a class of small, low-molecular-weight metal receptor proteins that are widely present in various biological cells and contain a Cu ion-binding domain.This domain is responsible for binding Cu ions and delivering them to specific transporters for Cu transport to individual organelles [198].Cu chaperones also play a role in preventing the toxic effects of free Cu ions on cells by inhibiting Cu binding to its intracellular components.For example, the expression level of antioxidant protein 1 (AtATX1) in Arabidopsis is upregulated by Cu-induced stress, and overexpression of AtATX1 significantly enhances the accumulation and tolerance of Arabidopsis to Cu [199].

Cu Uptake from Soil and Translocation to Shoots
The mechanism used to obtain Cu from the soil is similar to the reduced Fe absorption mechanism in dicotyledonous sand and non-grass monocotyledonous plants (Figure 2).For example, in Arabidopsis, roots are acidified by H + -ATPase, and Fe 3+ is reduced to soluble Fe 2+ using FRO2 localized to the plasma membrane [200].Fe 2+ is then absorbed by the high-affinity Fe transporter, IRT1, in the plasma membrane [201].Similar to Fe, Cu is reduced on the root surface of Arabidopsis by two Fe reductase enzymes, FRO4 and FRO5, which are activated in the absence of Cu [202].The reduced Cu + is then transported into the cell by the transporters AtCOPT1/2 and ZIP2/4 [203,204].AtCOPT1 is mainly expressed in root tips and localized to the plasma membrane, which mediates Cu uptake by roots from the soil.In the absence of Cu, the expression of AtCOPT1 is upregulated, enhancing the ability of roots to obtain Cu from the soil [203].During the absorption of Cu, OH is produced, which binds to non-selective cation channels on the plasma membrane and opens Ca ion channels.This process promotes the growth and development of roots [205].Similar to AtCOPT1, the expression of AtCOPT2 is also induced by Cu deficiency.Interestingly, when P is deficient in plants, AtCOPT2 transmits Cu to Cu-related proteins in response to low P signals by participating in P signal transduction [32].In the case of Cu restriction, the expression of ZIP2 and ZIP4 is induced, whereas that of ZIP4 is inhibited in the presence of excess Cu [206].However, recent studies have been partially contradictory because Cu has been reported to overregulate ZIP4 expression.In addition, because ZIP4 expression is also correlated with Zn levels, there may be crosstalk in plant absorption of Cu and Zn [25].
roots from the soil.In the absence of Cu, the expression of AtCOPT1 is upregulated, enhancing the ability of roots to obtain Cu from the soil [203].During the absorption of Cu, OH is produced, which binds to non-selective cation channels on the plasma membrane and opens Ca ion channels.This process promotes the growth and development of roots [205].Similar to AtCOPT1, the expression of AtCOPT2 is also induced by Cu deficiency.Interestingly, when P is deficient in plants, AtCOPT2 transmits Cu to Cu-related proteins in response to low P signals by participating in P signal transduction [32].In the case of Cu restriction, the expression of ZIP2 and ZIP4 is induced, whereas that of ZIP4 is inhibited in the presence of excess Cu [206].However, recent studies have been partially contradictory because Cu has been reported to overregulate ZIP4 expression.In addition, because ZIP4 expression is also correlated with Zn levels, there may be crosstalk in plant absorption of Cu and Zn [25].In monocotyledonous plants, most members of the COPT family have not been well studied except COPT7.The expressions of OsCOPT1, OsCOPT5, and OsCOPT7 were upregulated under Cu-deficient conditions.OsCOPT1 and OsCOPT5 synergistically mediate In monocotyledonous plants, most members of the COPT family have not been well studied except COPT7.The expressions of OsCOPT1, OsCOPT5, and OsCOPT7 were upregulated under Cu-deficient conditions.OsCOPT1 and OsCOPT5 synergistically mediate low-affinity Cu transport in yeast mpy17 mutants by interacting with another protein, XA13, in rice [207].These three proteins synergistically mediate Cu transport in rice.OsCOPT2, OsCOPT3, and OsCOPT4 physically interact with OsCOPT6, which can compensate for the absorption of Cu by the yeast mutants scctr1 and scctr3 [208].However, OsCOPT7 could also compensate for Cu absorption in the yeast mutants.Recently, it has been proposed that OsCOPT7 is a Cu-exporting protein found in the vacuoles and endoplasmic reticu-lum [33].It physically interacts with OsATX1 and plays an important role in regulating the root-to-shoot translocation of Cu in rice [33].
Once Cu is absorbed by the roots, it must be translocated to the shoot.In rice, OsHMA5, which is located in the plasma membrane, is responsible for the transport of Cu from the pericycle to the xylem through its interaction with OsATX1 [34].Mutants of oshma5 reduce the transport of Cu from roots to stems and seeds, consequently increasing the Cu concentration in roots [34].Furthermore, OsATX1 interacts with OsHMA4, OsHMA6, and OsHMA9 to control the root-to-shoot translocation of Cu and its delivery to the developing tissue of rice [199].Similarly, AtHMA5 is mainly expressed in the roots, and its expression is upregulated by excessive Cu.The athma5 mutants are more sensitive to Cu and accumulate higher levels of Cu in their roots.It has been suggested that athma5 plays a detoxification role in the roots by translocating Cu to the xylem [18].However, the tissue/organ-specific expression of AtATX1 and the patterns of Cu accumulation in different tissues and organs remain unknown.Therefore, it is difficult to determine whether AtATX1 affects Cu transport and distribution in plants through interactions with P 1B -type ATPase.
During long-distance transport of Cu in the vascular systems of dicotyledonous plants, Cu + may be oxidized to Cu 2+ to facilitate transportation to the aboveground parts.Cu 2+ binds to specific metal chelates in the xylem and phloem, such as metallothionein (MT), niacinamide, and 2 ′ -deoxylyconic acid (DMA) [209].To date, AtYSL2/AtYSL3 proteins have been well studied in vascular tissues.Both proteins are located on the lateral plasma membrane of the xylem parenchyma cells [23].AtYSL2 transports Cu-NA and Fe-NA complexes, whereas overexpression of AtYSL3 significantly increases the accumulation of Cu in leaves [210].In Arabidopsis, these two transporters are involved in the lateral movement of Cu in root and stem vascular bundles.In rice, it has been found that OsYSL16 is not only involved in Fe transport from the roots to the aboveground parts but is also responsible for transporting Cu-NA complexes in vegetative organs to new tissues and seeds through the phloem.This process facilitates the distribution of nutrients from vegetative organs to reproductive organs [211].It has been documented that the transport of Cu-NA from old leaves to new leaves and from flag leaves to spikelets is significantly reduced in osysl16 knockout lines.Moreover, additional Cu levels can enhance the germination rate of mutants.In Arabidopsis, a protein known as AtCOPT6 has also been reported to participate in Cu remobilization and redistribution from green tissues to reproductive organs [16].

Intracellular Transport of Cu
It has been reported that the cytoplasm of eukaryotic cells is essentially free of Cu; Cu ions are bound to metal partners and transferred to various Cu-dependent enzymes and different organelles (such as the mitochondria and chloroplasts) or sequestered in vacuoles, exoplasmic bodies, or expelled out of the cell to eliminate excess Cu in the cytoplasm [8] (Figure 2).

Vacuoles as Cu Stores
Vacuoles are the primary storage organelles in cells that store nutrients, including essential metals such as Cu, Zn, and Mn, and toxic elements, including nonessential metals such as cadmium and lead [212].Therefore, vacuoles play an important role in the maintenance of intracellular metal ion homeostasis through the sequestration of metal ions during cell detoxification and the influx of metal ions in the vacuoles under nutrientdeficient conditions.AtCOPT5 is a Cu transporter located in the vacuole membrane that transfers Cu stored in the vacuole to the cytoplasm to maintain normal plant growth under Cu-deficient conditions [15].In rice, OsHMA4 is involved in sequestering Cu in the vacuoles of root cells.Mutations in OsHMA4 result in increased Cu transport from the roots to the shoots, leading to increased Cu accumulation in the grains [212,213].Cu transporters in tonoplasts have also been reported in other species.For example, both CsHMA5.1 and CsHMA5.2mediate the transport of Cu from the cytoplasm to the vacuole.The transcript of CsHMA5.1 is root-specific and unaffected by Cu concentration, whereas the transcript of CsHMA5.2 is significantly upregulated in the presence of excess Cu [214].Under the toxic effects of Cu, CsHMA5.2 can increase the accumulation of Cu in the vacuoles of cucumber cells [214].

Cu Accumulation in Chloroplasts
Proteins in chloroplasts and mitochondria are involved in electron transport and redox processes, and Cu is required as a cofactor to perform these functions [215].In the chloroplast, Cu is transported to plastocyanin, a mobile electron carrier that plays an important role in the synthesis of photoreactive ATP and NADPH [57].In Arabidopsis, AtPCH1 and AtCCS are the main components responsible for Cu allocation to chloroplasts [216].AtPCH1 transfers Cu from the cytoplasm to the intermembrane space, and then the metal to AtPAA1/AtHMA6.It mediates the transport of Cu to the chloroplast matrix, where metal ions bind to AtCCS and are transferred to AtCSD2 and AtPAA2/AtHMA8, which are located in the thylakoid membrane [19,21].Finally, AtPAA2/AtHMA8 delivers Cu to plastocyanin in the thylakoid lumen.AtPAA2/AtHMA8 may also play a role in protecting thylakoids from Cu toxicity at high Cu concentrations [21].

Cu Accumulation in Mitochondria
Although Cu is also important for mitochondrial function, the transport mechanism of Cu in plant cell mitochondria remains unclear compared with that in chloroplasts.However, the transport mechanism of Cu in the mitochondria of yeast has been well studied.It has been shown that the Cu-chaperone ScCOX17 (cytochrome c oxidase) delivers Cu to the Cu-chaperone proteins ScCOX11 and ScSCO1 in the mitochondrial membrane, which then loads Cu to the CuA and CuB centers [217].They provide Cu for COX (Cudependent cytochrome c oxidase) subunits I and II, respectively.There are two homologues of ScCOX17 in Arabidopsis: AtCOX17-1 and AtCOX17-2.AtCOX17-1 is localized to the mitochondria and is involved in the transfer of Cu within the mitochondria [30].In addition, two homologs of SCO1 in yeast have been found in Arabidopsis: AtHCC1 and AtHCC2.Although the two ATHCCS share a high degree of homology, the CXXXC motif and histidine residue critical for Cu binding are not conserved in HCC2, suggesting that the two proteins have different roles.AtHCC1 is upregulated under excess Cu and contains CXXXC motifs and specific histidine residues required for Cu binding [218].In contrast, AtHCC2 lacks a Cu-binding motif and is therefore thought to be involved in redox regulation and the UV-B stress response rather than Cu transport within the mitochondria in plants [219].AtCOX11, a homolog of the yeast ScCOX11, has also been identified in Arabidopsis and is localized to the mitochondria.Interference and overexpression of AtCOX11 can affect COX activity and reduce pollen germination, indicating that COX11 plays a key role in Cu homeostasis in the mitochondria [220].

Cu Homeostasis in Endomembranes
In Arabidopsis, cytoplasmic Cu binds to the Cu-chaperone protein CCH, which interacts with AtHMA7/AtRAN1 located in the Golgi apparatus to transport Cu to the intracavity of the vesicles, where Cu is integrated into the ethylene receptor protein ETR1 [221].In Arabidopsis, the ETR1 receptor contains five subunits that mediate its response to ethylene.They consist of two domains: an N-terminal transmembrane domain, which is involved in Cu and ethylene binding, and a cytoplasmic C-terminal domain, which is involved in signal transduction [222].After binding to the transmembrane domain, Cu is required as a cofactor to deliver it to the receptor and thus bind ethylene with a high affinity [222].The transfer of Cu to ethylene receptors may be catalyzed by the ATPase AtHMA7/AtRAN1.Loss of function of AtRAN1/AtHMA7 in Arabidopsis leads to yellowing of seedlings and a constitutive ethylene reaction, resulting in severe growth inhibition or death [223].In addition to delivering Cu to proteins that require it, the secretory pathway-targeted HMA7/RAN1 transporter may also play a role in maintaining whole-cell Cu homeosta-sis, thereby enhancing plant tolerance to excess Cu [221].In addition to the P 1B -type Cu transport ATPase, members of the major facility superfamily (MFS) family are also involved in Golgi-mediated Cu homeostasis in plant cells.TaCT1 is a transporter from the common wheat MFS family that is located in the Golgi apparatus and participates in Cu transport.In contrast to white grass and AtRAN1/AtHMA7, the transcriptional levels of wheat TaCT1 were downregulated during excess Cu and upregulated during Cu deficiency.This suggests that Cu transporters in the MFS family in plants help maintain cellular Cu homeostasis by facilitating Cu uptake under Cu-deficient conditions [42].In addition to the Golgi apparatus, the endoplasmic reticulum also plays an important role in intracellular Cu homeostasis.Recently, it was proposed that OsCOPT7 is involved in transporting Cu from the endoplasmic reticulum to the cytoplasm.It physically interacts with OsATX1 to regulate the root-to-shoot translocation of Cu in rice [33].

Cu Homeostasis by Efflux Pathway
In the case of Cu poisoning, plant cells maintain Cu homeostasis in the cytoplasm, primarily by expelling Cu 2+ from the plasma membrane.AtHMA5 interacts with the Cu partner ATX1-like to transport excess Cu to the plasma membrane, where it is excreted.AtHMA5 can transport Cu to exosomal oxidase and laccase, both of which are involved in cell wall formation [18].In rice, OsHMA5 and OsHMA9 participate in Cu expulsion and reduce the accumulation of Cu in cells [35].In addition, BnPCR10.1 from Brassica napus and SlPCR6 from Salix linearistipularis enhance Cu efflux from cells [44,224].

Cu Homeostasis Is Mediated by the Regulation of Transcription Factors
In recent years, the significance of transcription factors in maintaining Cu homeostasis has been gradually revealed.SPL7 (SQUAMOSA promoter binding protein-like 7) is a well-known core transcription factor involved in sensing Cu deficiency signals [225,226].Under Cu deficiency conditions, SPL7 can bind to the GTAC motif within the Cu response element in the promoter region of Cu-responsive genes.This binding activates the Cumetal reductase FRO4/5 and the Cu transporter COPT1/2/6 expression of a high-affinity Cu uptake system [202,206,226,227].Some evidence suggests that SPL7 needs to directly interact with multiple proteins, such as KIN17, HY5 (elongated hypocotyl 5), and CITF1 (Cu-deficiency-induced transcription factor), to promote its binding to the Cu response element of the target gene [99,227,228].Furthermore, under conditions of Cu deficiency, SPL7 inhibits the expression of other Cu-responsive genes by activating miRNAs to deliver Cu to the essential Cu-containing proteins.These miRNAs are called Cu-miRNAs and include miR397, miR398, miR408, and miR857 [105,229].They target the mRNA of less important Cu-containing proteins, such as Cu/Zn superoxide dismutase and plantain cyanin.Evidence suggests that Cu homeostasis in plant cells is regulated by circadian rhythms.It has been proposed that SPL7 regulates the expression of COPT2 and Fe superoxide dismutase 1 (FSD1) through circadian rhythms in response to cellular demands for Cu [230] (Figure 3).In addition, the circadian clock protein TCP16 (Teosinte branched 1, cycloidea proliferation cell factor 1) can bind to the COPT3 promoter and down-regulate its expression, thereby reducing the Cu content and affecting pollen development [231] (Figure 3).Other well-known transcription factor families, such as WRKY, are also directly involved in regulating cellular Cu homeostasis.Recent studies have shown that it can induce transcription of OsWRKY37, which binds to promoters of OsCOPT6 and OsYSL16, thereby positively regulating their expression [232] (Figure 3).However, some transcription factors affect Cu absorption and distribution indirectly.FIT (Fer-like iron deficiencyinduced transcription factor) and four bHLH Ib transcription factors (bHLH38, bHLH39, bHLH100, and bHLH101) directly bind to the promoters of COPT2, FRO4, and FRO5 to enhance Cu absorption and alleviate Fe-deficiency symptoms, contributing to the mutual balance between Cu and Fe [233].PIF3/4/5 (phytochrome-interacting factors) directly binds to the promoters of miR408 to regulate dark-induced senescence in Arabidopsis leaves by facilitating intracellular Cu redistribution [234].The findings highlight the enhancement of Cu uptake by plants and their subsequent transportation to the soil and reproductive organs, thereby promoting crop growth and increasing crop yield.
tion factors affect Cu absorption and distribution indirectly.FIT (Fer-like iron deficiencyinduced transcription factor) and four bHLH Ib transcription factors (bHLH38, bHLH39, bHLH100, and bHLH101) directly bind to the promoters of COPT2, FRO4, and FRO5 to enhance Cu absorption and alleviate Fe-deficiency symptoms, contributing to the mutual balance between Cu and Fe [233].PIF3/4/5 (phytochrome-interacting factors) directly binds to the promoters of miR408 to regulate dark-induced senescence in Arabidopsis leaves by facilitating intracellular Cu redistribution [234].The findings highlight the enhancement of Cu uptake by plants and their subsequent transportation to the soil and reproductive organs, thereby promoting crop growth and increasing crop yield.

Conclusions and Future Perspective
The relevance of Cu as a plant micronutrient remains largely underestimated.It is often believed that plant cells have low physiological requirements for Cu and that they absorb Cu in amounts that meet their metabolic requirements.However, in natural and agricultural environments, Cu availability is limited by factors such as soil type and its physical and chemical properties.Undetected and unrepaired latent Cu deficiencies can impair plant growth and hinder sustainable and productive agriculture.Cu deficiency can be remedied by applying Cu-based fertilizers, which may lead to Cu accumulation in the soil.However, with industrial production (such as mineral development and chemical manufacturing) and agricultural activities (such as the use of Cu-based agricultural chemicals), soil Cu pollution has become increasingly serious.Excessive Cu accumulation in cells inhibits plant root growth and leaf photosynthesis, thereby severely limiting plant productivity.Several measures have been used to mitigate the toxicity of Cu in soil to plants, including the application of soil amendments such as lime or fly ash, adding organic fertilizers, and limiting the use of Cu-based pesticides.However, the choice and availability of these remedies depend on various factors, including availability, cost, and duration of effectiveness.

Conclusions and Future Perspective
The relevance of Cu as a plant micronutrient remains largely underestimated.It is often believed that plant cells have low physiological requirements for Cu and that they absorb Cu in amounts that meet their metabolic requirements.However, in natural and agricultural environments, Cu availability is limited by factors such as soil type and its physical and chemical properties.Undetected and unrepaired latent Cu deficiencies can impair plant growth and hinder sustainable and productive agriculture.Cu deficiency can be remedied by applying Cu-based fertilizers, which may lead to Cu accumulation in the soil.However, with industrial production (such as mineral development and chemical manufacturing) and agricultural activities (such as the use of Cu-based agricultural chemicals), soil Cu pollution has become increasingly serious.Excessive Cu accumulation in cells inhibits plant root growth and leaf photosynthesis, thereby severely limiting plant productivity.Several measures have been used to mitigate the toxicity of Cu in soil to plants, including the application of soil amendments such as lime or fly ash, adding organic fertilizers, and limiting the use of Cu-based pesticides.However, the choice and availability of these remedies depend on various factors, including availability, cost, and duration of effectiveness.
Increasing evidence suggests that using the endogenous metal transport mechanism in plants could be a key measure to address issues related to metal deficiency or toxicity.For example, through gene editing, the functions of high-affinity Cu uptake and transport proteins in plant roots are suppressed to reduce Cu uptake by plants growing in Cu-polluted areas.Others improve plant tolerance to metals by sequestering metal ions into cellular compartments such as vacuoles.However, a prerequisite for implementing these measures is to fully understand the regulatory mechanisms of Cu transport between tissues and cellular homeostasis.Key questions include, for example, what is the complete molecular pathway through which Cu, taken up by roots from the soil, is transported to the vascular bundles and loaded or unloaded in the xylem or phloem?In grasses, such as rice, certain nodes play a role in the transport of metal ions from roots to aboveground parts and the redistribution of aboveground parts.Given that the aboveground parts of plants have a limited demand for Cu, how do these nodes precisely regulate Cu transport?What transport proteins are involved in these processes?Furthermore, at the cellular level, is the trans-Golgi network-mediated vesicle secretion mechanism involved in intracellular Cu detoxification, in addition to vacuolar sequestration?
Other challenges to be solved include plant sensing and signaling of Cu status.This review highlighted some transcription factors, such as SPL7 and OsWRKY37, that regulate the expression of related genes in response to Cu deficiency in plants.However, the signaling mechanisms underlying the response of plants to Cu stress remain unclear.
Future research should prioritize addressing these scientific questions and investigating the regulatory mechanisms of plants in response to Cu deficiency or stress.Because Cu deficiency and toxicity are both limiting factors for agricultural productivity, a better understanding of Cu homeostasis in plants, combined with related biological breeding, will enhance the Cu utilization efficiency and/or Cu tolerance of crops in alkaline or Cu-contaminated soils in the future.

Figure 1 .
Figure 1.Overview of Cu sources, soil bioavailability, and the effects of its deficiency and excess on plant systems (for details, see text).

Figure 1 .
Figure 1.Overview of Cu sources, soil bioavailability, and the effects of its deficiency and excess on plant systems (for details, see text).

Figure 2 .
Figure 2. Overview of Cu transport mechanisms in plants.(A) Tissue specificity of Cu transporters, chaperones, and complexes.(B,C) subcellular localization of Cu transporters, chaperones, and complexes in root or leaf cells.In (B,C), black arrows indicate import into the cytosol, and purple arrows indicate export out of the cytosol.Proteins that undergo subcellular localization are only shown in yeast but not in plants and are indicated by a question mark.ZIP, Zn-regulated transporter; COPT, Copper transporter protein; FRO, Ferric reductase oxidase; HMA, Heavy metal ATPase; PCR, Plant cadmium resistance; PCH, Plas-tid chaperone; ATX1, Antioxidant protein; COX, Cytochrome coxidase; PCs, Phytochelatins; CCS/CCH, Cu chaperone; MTs, Metallothioneins; HCC1, the Arabidopsis homolog of the yeast mitochondrial copper chaperone SCO1; Pc, Plastocyanin; CT, MFS-type Cu trans-porter; YSL, Yellow stripe like; HIPP16, Heavy metal-associated isoprenylated plant protein 16.

Figure 2 .
Figure 2. Overview of Cu transport mechanisms in plants.(A) Tissue specificity of Cu transporters, chaperones, and complexes.(B,C) subcellular localization of Cu transporters, chaperones, and complexes in root or leaf cells.In (B,C), black arrows indicate import into the cytosol, and purple arrows indicate export out of the cytosol.Proteins that undergo subcellular localization are only shown in yeast but not in plants and are indicated by a question mark.ZIP, Zn-regulated transporter; COPT, Copper transporter protein; FRO, Ferric reductase oxidase; HMA, Heavy metal ATPase; PCR, Plant cadmium resistance; PCH, Plas-tid chaperone; ATX1, Antioxidant protein; COX, Cytochrome coxidase; PCs, Phytochelatins; CCS/CCH, Cu chaperone; MTs, Metallothioneins; HCC1, the Arabidopsis homolog of the yeast mitochondrial copper chaperone SCO1; Pc, Plastocyanin; CT, MFS-type Cu trans-porter; YSL, Yellow stripe like; HIPP16, Heavy metal-associated isoprenylated plant protein 16.